Influenza A viruses (IAVs) are enveloped viruses that belong to the Orthomyxoviridae family, and contain a genome comprised of eight single-stranded negative-sense RNA viral segments that encode for 10-14 proteins (Baker et al., 2015, Future Virology, 10: 715-730). The hemagglutinin (HA) and the neuraminidase (NA) glycoproteins are the major antigenic determinants of IAV and are essential for receptor binding and fusion, and virion release, respectively (Varghese et al., 1992, Proteins, 14:-327-332). IAV HA and NA glycoproteins within infected organisms and populations are driven to evolve antigenic variants via immunologic pressure, and positive selection of fit viruses occurs gradually in a process known as antigenic drift (Carrat et al., 2007, Vaccine, 24: 6852-6862). The antigenic diversity of glycoproteins is used to further classify IAVs, of which there are 18 HA and 11 NA subtypes (Palese, 2007, The Viruses and Their Replication, Fields Virology, 5th ed. Lippincott Williams and Wilkins; Tong et al., 2013, PLoS, pathogens, e1003657). In addition, antigenically distinct isolates can also exist within the same subtype, referred to as drifted variants. IAVs exist mainly in the wild aquatic fowl reservoir (de Jong et al., 2007, J Virol, 81: 4315-4322; Taubenberger and Kash, 2010, Cell Host & Microbe, 7: 440-451; Webster et al., 1992, Microbiological Reviews, 56: 152-179; Yoon et al., 2014, Current Topics in Microbiology and Immunology, 385: 359-375) and only a small number of mammalian hosts are currently recognized to sustain transmission and sustention of IAVs.
Canine influenza or dog flu is a common and contagious respiratory disease of dogs caused by two IAVs: the H3N8 equine-origin influenza virus that transferred to dogs in the United States around 1999 (Crawford et al., 2005, Science, 310: 482-485); and the avian virus-like H3N2 that transferred to dogs in Asia around 2005 (Song et al., 2008, Emerging Infectious Diseases, 14: 741-746). Recently, in 2015, an outbreak of H3N2 canine influenza virus (CIV) similar to the ones detected in dogs in Asia, was reported in the United States (2015, Javrna-J Am Vet med A, 246: 1049). Notably, H3N2 CIV seems to have a broad host range, as it has been isolated from cats during an outbreak of respiratory disease in a shelter in South Korea (Jeoung et al., 2013, Veterinary Microbiology, 165: 281-286; Song et al., 2011, The Journal of General Virology, 92: 2350-2355). CIV represents a new threat to canine health in the United States and worldwide, as the virus rapidly spreads to dogs throughout the racing track circuit (Crawford et al., 2005, Science, 310: 482-485; Yoon et al., 2005, Emerging Infectious Diseases, 11: 1974-1976) or animal shelters Crawford et al., 2005, Science, 310: 482-485; Holt et al., 2010, Journal of the American Veterinary Medical Association, 237: 71-73; Pecoraro et al., 2013, Journal of Veterinary Diagnostic Investigation, 25: 402-406). CIV is a relatively new virus and almost all dogs are susceptible to infection when they are newly exposed because they have not natural immunity. Most dogs that develop CIV infection have a mild illness, but some dogs get very sick and require treatment (Gonzalez et al., 2014, J Virol, 88: 9208-9219). The recent emergence of CIV has important implications, because the ecological niche of IAVs has increased significantly and both of these CIVs (H3N8 and H3N2) have continuously circulated in the dog population since they emerged, creating many opportunities for exposure in humans and other species. Importantly, as dogs are susceptible to mammalian (equine-origin H3N8 CIV) and avian (avian-origin H3N2 CIV) IAVs, they possess all the attributes to become, like pigs, “mixing vessel” species for the emergency of new IAVs with pandemic potential for humans. The fact that dogs are the closest human companion animals makes reassortment between canine and human viruses more likely to occur. In fact, it has been shown that reassortments between H3N8 or H3N2 CIVs and human IAVs are feasible (Song et al., 2015, The Journal of General Virology, 96: 254-258; Song et al., The Journal of General Virology, 93: 551-554). Hence, society should be alert to the possible transmission and potential emergence of CIVs in humans. This is particularly alarming, because the introduction of novel, antigenically distinct glycoproteins (HA and NA) within the backbone of human IAVs has previously been associated with human pandemics (Yen et al., 2009, Current topics in microbiology and immunology, 333: 3-24).
Vaccination is universally accepted as the most effective strategy for the prevention of influenza viral infections (Pica et al., 2013, Annual Review of Medicine, 64: 189-202; Wong et al., 2013, Clinical Microbiology Reviews, 26: 476-492). To date, three types of influenza virus vaccines have been approved by the United States FDA for human use: recombinant viral HA, inactivated influenza vaccines (IIVs), and live attenuated influenza vaccines (LAIVs) (Pica et al., 2013, Annual Review of Medicine, 64: 189-202; Belshe et al., 2007, The New England Journal of Medicine, 356: 685-696; Cox et al., 2008, Influenza and other Respiratory Viruses: 2: 211-219; Osterholm et al., 2012, The Lancet Infectious Diseases, 12: 36-44; Pronker et al., 2012, Vaccine, 30: 7344-7347). In dogs, IIV against H3N8 (and recently H3N2) CIVs are commercially available. IIVs are administered intramuscularly and elicit protective humoral immunity by inducing the production of neutralizing antibodies that target epitopes on HA, typically proximal to the receptor binding site (Osterholm et al., 2012, The Lancet Infectious Diseases, 12: 36-44; Belongia et al., 2009, Journal of Infectious Diseases, 199: 159-167) that prevent (neutralize) viral infection. On the other hand, LAIV mimics the natural route of viral infection and are able to elicit more efficient cellular and humoral immune responses (Belshe et al., 2007, The New England Journal of Medicine, 356: 685-696), providing better immunogenicity and protection against both homologous and heterologous influenza virus strains (Pica et al., 2013, Annual Review of Medicine, 64: 189-202; Gorse et al., 1991, Chest, 100: 977-984).
In 2006, the American Veterinary Medical Association (AVMA) called for the urgent development of an effective vaccine against CIV. A vaccine made from inactivated virus have been developed that is administered subcutaneously as two doses to reduce the severity of the CIV disease and to reduce the incidence of CIV infection in naive dogs (Nobivac, Merck). However, to date, no LAIV for CIV infections has been developed. Thus there is a need in the art for improved vaccines for CIV. The present invention satisfies this unmet need.